Enzymatic catalysis is one of the fundamental processes that drives the dynamic landscape of post-translational modifications (PTMs), expanding the structural and functional diversity of proteins. Here, we assessed enzyme specificity using a top-down ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS) workflow. We successfully applied trapped IMS (TIMS) to investigate site-specific N-ε-acetylation of lysine residues of full-length histone H4 catalyzed by histone lysine acetyltransferase KAT8. We demonstrate that KAT8 exhibits a preference for N-ε-acetylation of residue K16, while also adding acetyl groups on residues K5 and K8 as the first degree of acetylation. Achieving TIMS resolving power values of up to 300, we fully separated mono-acetylated regioisomers (H4K5ac, H4K8ac, and H4K16ac). Each of these separated regioisomers produce unique MS/MS fragment ions, enabling estimation of their individual mobility distributions and the exact localization of the N-ε-acetylation sites. This study highlights the potential of top-down TIMS-MS/MS for conducting enzymatic assays at the intact protein level and, more generally, for separation and identification of intact isomeric proteoforms and precise PTM localization.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1002/pmic.202200471 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Emerging opportunities for intact and native protein analysis using chemical proteomics.
Anal Chim Acta
February 2025
Department of Chemistry, University of Texas at Austin, Austin, TX, 78712, United States. Electronic address:
Chemical proteomics has advanced small molecule ligand discovery by providing insights into protein-ligand binding mechanism and enabling medicinal chemistry optimization of protein selectivity on a global scale. Mass spectrometry is the predominant analytical method for chemoproteomics, and various approaches have been deployed to investigate and target a rapidly growing number of protein classes and biological systems. Two methods, intact mass analysis (IMA) and top-down proteomics (TDMS), have gained interest in recent years due to advancements in high resolution mass spectrometry instrumentation.
View Article and Find Full Text PDFCarving Metal-Organic-Framework Glass Based Solid-State Electrolyte Via a Top-Down Strategy for Lithium-Metal Battery.
Angew Chem Int Ed Engl
January 2025
KU Leuven, Materials engineering, Kasteelpark Arenberg 44 bus 2450, 3001 LEUVEN Belgium, LEUVEN, BELGIUM.
Traditional polymer solid electrolytes (PSEs) suffer from low Li conductivity, poor kinetics and safety concerns. Here, we present a novel porous MOF glass gelled polymer electrolyte (PMG-GPE) prepared via a top-down strategy, which features a unique three-dimensional interconnected graded-aperture structure for efficient ion transport. Comprehensive analyses, including time-of-flight secondary ion mass spectrometry (TOF-SIMS), Solid-state 7Li magic-angle-spinning nuclear magnetic resonance (MAS-NMR), Molecular Dynamics (MD) simulations, and electrochemical tests, quantify the pore structures, revealing their relationship with ion conductivity that increases and then decreases as macropore proportion rises.
View Article and Find Full Text PDFRevealing Unpredicted Aspartic Acid Isomerization Hotspots by Probing Diagnostic Fragmentation Propensities in Top-Down and Middle-Down Mass Spectrometry.
J Am Soc Mass Spectrom
January 2025
Analytical Characterization, Biologics Analytical Development, Technical Research & Development, Novartis Pharma AG, WKL693.3.20, Postfach, CH-4002 Basel, Switzerland.
Isomerization of aspartic acid residues is a relevant degradation pathway of protein biopharmaceuticals as it can impair their biological activity. However, the in silico prediction of isomerization hotspots and their consequences remains ambiguous and misleading. We have previously shown that all ion differential analysis (AiDA) of middle-down spectra can be used to reveal diagnostic terminal and internal fragments with more sensitivity than the conventional fragment ion mass matching methodology.
View Article and Find Full Text PDFPEPPI-MS: gel-based sample pre-fractionation for deep top-down and middle-down proteomics.
Nat Protoc
January 2025
Advanced Research Support Center, Ehime University, Ehime, Japan.
Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS-PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics.
View Article and Find Full Text PDFProgress in Tandem Mass Spectrometry Data Analysis for Nucleic Acids.
Mass Spectrom Rev
January 2025
Department of Chemistry, University of Texas at Austin, Austin, Texas, USA.
Mass spectrometry (MS) has become a critical tool in the characterization of covalently modified nucleic acids. Well-developed bottom-up approaches, where nucleic acids are digested with an endonuclease and the resulting oligonucleotides are separated before MS and MS/MS analysis, provide substantial insight into modified nucleotides in biological and synthetic nucleic. Top-down MS presents an alternative approach where the entire nucleic acid molecule is introduced to the mass spectrometer intact and then fragmented by MS/MS.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!